IGF-1 receptor interacting proteins

ABSTRACT

The invention comprises a nucleic acid molecule with the sequence SEQ ID NO:5 and the complementary sequence, and its use in diagnosis and therapy. This nucleic acid molecule (IIP-10) is a gene which encodes an IGF-1 receptor binding polypeptide.

This is a divisional of application Ser. No. 09/453,195, filed Dec. 2,1999, now U.S. Pat. No. 6,368,826.

BACKGROUND OF THE INVENTION

The IGF-1 receptor signaling system plays an important role in tumorproliferation and survival and is implicated in inhibition of tumorapoptosis. In addition and independent of its mitogenic properties,IGF-1R activation can protect against or at least retard programmed celldeath in vitro and in vivo (Harrington et al., EMBO J. 13 (1994)3286-3295; Sell et al., Cancer Res. 55 (1995) 303-305; Singleton et al.,Cancer Res. 56 (1996) 4522-4529). A decrease in the level of IGF-1Rbelow wild type levels was also shown to cause massive apoptosis oftumor cells in vivo (Resnicoff et al., Cancer Res. 55 (1995) 2463-2469;Resnicoff et al., Cancer Res. 55 (1995) 3739-3741). Overexpression ofeither ligand (IGF) and/or the receptor is a feature of various tumorcell lines and can lead to tumor formation in animal models.Overexpression of human IGF-1R resulted in ligand-dependentanchorage-independent growth of NIH 3T3 or Rat-1 fibroblasts andinoculation of these cells caused a rapid tumor formation in nude mice(Kaleko et al., Mol. Cell. Biol. 10 (1990) 464-473; Prager et al., Proc.Natl. Acad. Sci. USA 91 (1994) 2181-2185). Transgenic miceoverexpressing IGF-II specifically in the mammary gland develop mammaryadenocarcinoma (Bates et al., Br. J. Cancer 72 (1995) 1189-1193) andtransgenic mice overexpressing IGF-II under the control of a moregeneral promoter develop an elevated number and wide spectrum of tumortypes (Rogler et al., J. Biol. Chem. 269 (1994) 13779-13784). Oneexample among many for human tumors overexpressing IGF-I or IGF-II atvery high frequency (>80%) are Small Cell Lung Carcinomas (Quinn et al.,J. Biol. Chem. 271 (1996) 11477-11483). Signaling by the IGF systemseems to be also required for the transforming activity of certainoncogenes. Fetal fibroblasts with a disruption of the IGF-1R gene cannotbe transformed by the SV40 T antigen, activated Ha-ras, or a combinationof both (Sell et al., Proc. Natl. Acad. Sci USA 90 (1993) 11217-11221;Sell et al., Mol. Cell. Biol. 14 (1994) 3604-3612), and the E5 proteinof the bovine papilloma virus is also no longer transforming (Morrioneet al., J. Virol. 69 (1995) 5300-5303). Interference with the IGF/IGF-1Rsystem was also shown to reverse the transformed phenotype and toinhibit tumor growth (Trojan et al., Science 259 (1993) 94-97; Kalebicet al., Cancer Res. 54 (1994) 5531-5534; Prager et al., Proc. Natl.Acad, Sci. USA 91 (1994) 2181-2185; Resnicoffet al., Cancer Res. 54(1994) 2218-2222; Resnicoff et al., Cancer Res. 54 (1994) 4848-4850;Resnicoff et al., Cancer Res. 55 (1995) 2463-2469. For example, miceinjected with rat prostate adenocarcinoma cells (PA-III) transfectedwith IGF-1R antisense cDNA (729 bp) develop tumors 90% smaller thancontrols or remained tumor-free after 60 days of observation (Burfeindet al., Proc. Natl. Acad. Sci. USA 93 (1996) 7263-7268). IGF-1R mediatedprotection against apoptosis is independent of de-novo gene expressionand protein synthesis. Thus, IGF-1 likely exerts its anti-apoptoticfunction via the activation of preformed cytosolic mediators.

Some signaling substrates which bind to the IGF-1R (e.g. IRS-1, SHC, p85P13 kinase etc., for details see below) have been described. However,none of these transducers is unique to the IGF-1R and thus could beexclusively responsible for the unique biological features of the IGF-1Rcompared to other receptor tyrosine kinase including the insulinreceptor. This indicates that specific targets of the IGF-1R (or atleast the IGF-receptor subfamily) might exist which trigger survival andcounteract apoptosis and thus are prime pharmaceutical targets foranti-cancer therapy.

By using the yeast two-hybrid system it was shown that p85, theregulatory domain of phosphatidyl inositol 3 kinase (PI3K), interactswith the IGF-1R (Lamothe, B., et al., FEBS Lett. 373 (1995) 51-55;Tartare-Decker, S., et al., Endocrinology 137 (1996) 1019-1024). Howeverbinding of p85 to many other receptor tyrosine kinases of virtually allfamilies is also seen. Another binding partner of the IGF-1R defined bytwo-hybrid screening is SHC which binds also to other tyrosine kinasessuch as trk, met, EGF-R and the insulin receptor (Tartare-Deckert, S.,et al., J. Biol. Chem. 270 (1995) 23456-23460).

The insulin receptor substrate 1 (IRS-1) and insulin receptor substrate2 (IRS-2) were also found to both interact with the IGF-1R as well asthe insulin receptor (Tartare-Deckert, S., et al., J. Biol. Chem. 270(1995) 23456-23460; He, W., et al., J. Biol. Chem. 271 (1996)11641-11645; Dey, R. B., et al., Mol. Endocrinol. 10 (1996) 631-641).Grb 10 which interacts with the IGF-1R also shares many tyrosine kinasesas binding partners, e.g. met, insulin receptor, kit and abl (Dey, R.B., et al., Mol. Endocrinol. 10 (1996) 631-641; Morrione, A., et al.,Cancer Res. 56 (1996) 3165-3167). The phosphatase PTP1D (syp) shows alsoa very promiscuous binding capacity, i.e. binds to IGF-1R, insulinreceptor, met and others (Rocchi, S., et al., Endocrinology 137 (1996)4944-4952). More recently, mSH2-B and vav were described as binders ofthe IGF-1R, but interaction is also seen with other tyrosine kinases,e.g. mSH2-B also bind to ret and the insulin receptor (Wang, J., andRiedel, H., J. Biol. Chem. 273 (1998) 3136-3139). Taken together, the sofar described IGF-1R binding proteins represent relatively unspecifictargets for therapeutic approaches, or are in the case of the insulinreceptor substrates (IRS-1, IRS-2) indispensable for insulin-drivenactivities.

SUMMARY OF THE INVENTION

The present invention relates to IGF-1 receptor interacting proteins(IIPs); nucleic acids coding therefor; and their use for diagnostics andtherapeutics, especially in the field of cancer. In particular, theinvention relates to the identification of said genes in mammaliancells, especially in malignant tumor cells; to gene therapy methods forinhibiting the interaction between IGF-1 receptor and IIPs; methods ofscreening for potential cancer therapy agents; and cell lines and animalmodels useful in screening for and evaluating potentially usefulpharmaceutical agents that inhibit the interaction between IIiPs andIGF-1 receptor. The present invention relates in particular to thecloning and characterization of the gene IIP-11 and the gene productsthereof. Said gene products (polypeptides, mRNA) are especiallycharacterized as having the ability to modulate the IGF-1 receptorsignaling pathway. The function of the gene products according to theinvention is therefore to modulate signal transduction of the IGF-1receptor. Forced activation of IIPs therefore correlates with increasedtumor cell proliferation, survival and escape of apoptosis. It is anobject of the invention to provide novel genes encoding binding proteinsof IGF-1R as well as the corresponding polypeptides which modulate,preferably activate the IGF-1 receptor signaling pathway. It isenvisioned that this invention provides a basis for new cancer therapiesbased on the modulation, preferably inhibition, of the interactionbetween IGF-1R and IIPs.

DESCRIPTION OF THE FIGURES AND SEQUENCES

FIG. 1 Domain structure of yeast two-hybrid baits which were used toscreen cDNA libraries for cytoplasmic binding proteins of the IGF-1receptor. The LexA DNA binding domain was fused to the cytoplasmic (cp)domain (nt 2923 to 4154) of the wildtype IGF-1 receptor (a) or thekinase inactive mutant (K/A mutation at aa 1003) (b) (Ullrich, A., etal., EMBO J. 5 (1986) 2503-2512; Weidner, K. M., et al., Nature 384(1996) 173-176). The nucleotide and amino acid sequence of two differentlinkers inserted between the LexA DNA-binding domain and the receptordomain are shown below. The I1a(wt IGF-1 receptor) and K1 (kinaseinactive mutant IGF-1 receptor) constructs contain an additional prolineand glycine compared to the I2 and K2 constructs.

FIG. 2 Modification of the yeast two-hybrid LexA/IGF-1 receptor baitconstruct.

-   -   a) Schematic illustration of cytoplasmic binding sites of the        IGF-1 receptor. The α-subunits of the IGF-1 receptor are linked        to the β-chains via disulfid bonds. The cytoplasmic part of the        β-chain contains binding sites for substrates in the        juxtamembrane and C-terminal domain.    -   b) Domain structure of the two-hybrid bait containing only the        juxtamembrane IGF-1 receptor binding sites. The juxtamembrane        domain of the IGF-1 receptor (nt 2923 to 3051) (Ullrich, A., et        al., EMBO J. 5 (1986) 2503-2512) was fused to the kinase domain        of tprmet (nt 3456 to 4229) (GenBank accession number:        HSU19348).    -   c) Domain structure of the two-hybrid bait containing only the        C-terminal IGF-1 receptor binding sites. The C-terminal domain        of the IGF-1 receptor (nt 3823 to 4149) (Ullrich, A., et al.,        EMBO J. 5 (1986) 2503-2512) was fused to the kinase domain of        tprmet (nt 3456 to 4229) (GenBank accession number: HSU19348).

FIG. 3 Isoforms of IIP-1.

-   -   a) Delineation of the cDNA sequences of IIP-1 and IIP-1 (p26).        Nucleotides are numbered above. The potential translation        initiation site within the IIP-1 cDNA is at position 63. The        first ATG as potential translation initiation site in the        alternative splice variant IIP-1 (p26) is at position 353. Both        cDNAs contain a stop codon at position 1062.    -   b) Domain structure of IIP-1 and IIP-1 (p26). Amino acid        positions are indicated above. In comparison to IIP-1 (p26)        IIP-1 contain additional 97 amino acids at the N-terminus. Both        isoforms of IIP-1 contain a PDZ domain spanning a region between        amino acids 129 and 213.

FIG. 4 Delineation of the IGF-1 receptor binding domain of IIP-1.Full-length IIP-1, its partial cDNA clones (IIP-1a and IIP-1b) anddeletion mutants (IIP-1a/mu1, IIP-1a/mu2, IIP-1a/mu3, IIP-1b/mu1) wereexamined for interaction with the IGF-1 receptor in the yeast two-hybridsystem. Yeast cells were cotransfected with a LexA IGF-1 receptor fusionconstruct and an activation plasmid coding for IIP-1 or the differentIIP-1 mutants fused to the VP16 activation domain. Interaction betweenIIP-1 or its mutants and the IGF-1 receptor was analyzed by monitoringgrowth of yeast transfectants plated out on histidine deficient mediumand incubated for 6d at 30° C. (diameter of yeast colonies: +++, >1 mmin 2d; ++, >1 mm in 4d; +, >1 mm in 6d; −, no detected growth). The PDZdomain can be defined as essential and sufficient for mediating theinteraction with the IGF-1 receptor. Nucleotide positions with respectto full length IIP-1 are indicated above.

FIG. 5 Protein sequence motifs of IIP-10. The amino acid sequence ofIIP-10 was analyzed using the computer program “Motifs” (WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis.)which looks for protein motifs by searching protein sequences forregular expression patterns described in the PROSITE Dictionary.

SEQ ID NO:1 Nucleotide sequence of IIP-1 (cDNA).

SEQ ID NO:2 Predicted amino acid sequence of IIP-1.

SEQ ID NO:3 Nucleotide sequence of the IIP-6 partial cDNA clone.

SEQ ID NO:4 Deduced amino acid sequence of the IIP-6 partial cDNA clone.Cysteine and histidine residues of the two Cys₂His₂ Zinc finger domainsare amino acids 72, 75, 88, 92, 100, 103, 116, and 120.

SEQ ID NO:5 Nucleotide sequence of IIP-10 (cDNA).

SEQ ID NO:6 Deduced amino acid sequence of IIP-10.

SEQ ID NO:7 Primer TIP2c-s.

SEQ ID NO:8 Primer TIP2b-r.

SEQ ID NO:9 Primer Hcthy-s.

SEQ ID NO:10 Primer Hcthy-r.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to IGF-1 receptor interacting proteins(IIPs); nucleic acids coding therefor; and their use for diagnostics andtherapeutics, especially in the field of cancer. The inventionpreferably comprises a nucleic acid encoding a protein (IIP-10) bindingto IGF-1 receptor selected from the group comprising

-   a) the nucleic acids shown in SEQ ID NO:5 or a nucleic acid sequence    which is complementary thereto,-   b) nucleic acids which hybridize under stringent conditions with one    of the nucleic acids from a) which encode a polypeptide showing at    least 75% homology with the polypeptide of SEQ ID NO:6 or-   c) sequences that, due to the degeneracy of the genetic code, encode    IIP-10 polypeptides having the amino acid sequence of the    polypeptides encoded by the sequences of a) and b).

The cDNA of IIP-10 codes for a new protein of 226 amino acid with acalculated molecular weight of 25.697. IIP-10 is a lysine rich protein(11%). IIP-10 contains an N-glycosylation site, several N-myristoylationsites, Ck2 and PKC phosphorylation sites, one tyrosine kinasephosphorylation site and one putative nuclear localization signal (FIG.5). The cDNA sequence of IIP-10 shows 65% homology to the cDNA sequenceof the Gallus Gallus thymocyte protein cthy28kD (EMBL accession number:GG34350). The amino acid sequences of IIP-10 and cthy28kD show 70%identity. Nt 383 to nt 584 of the IIP-10 cDNA are 94% identical to apartial cDNA described in WO 95/14772 (human gene signature HUMGS06271;accession number T24253). By immunofluorescence, flag-tagged IIP-10shows both a cytoplasmic and a nuclear localization in NIH3T3 cellsoverexpressing the IGF-1 receptor. Further yeast two-hybrid analysisrevealed that IIP-10 interacts in a phosphorylation dependent mannerwith the IGF-1 receptor. IIP-10 does not interact with the insulinreceptor. Deletion analysis of IIP-10 revealed that aa 19 to aa 226 aresufficient for binding to the IGF-1 receptor.

“Interaction” or “binding between IIP10 and the IGF-1 receptor” means aspecific binding of the IIP10 polypeptide to the IGF-1 receptor but notto control proteins such as lamin in the yeast two hybrid system.Specific binding to the IGF-1 receptor can be demonstrated usingglutathion-S-transferase (GST)-IIP fusion proteins expressed in bacteriaand IGF-1 receptors expressed in mammalian cells. Furthermore, anassociation between a Flag tagged IIP-10 fusion protein (cf. Weidner, K.M. et al., Nature 384 (1996) 173-176) and the IGF-1 receptor can bemonitored in mammalian cell systems. For this purpose eukaryoticexpression vectors are used to transfect the respective cDNAs.Interaction between the proteins is visualized by coimmunoprecipitationexperiments or subcellular localization studies using anti-Flag oranti-IGF-1 receptor antibodies. Further provided by the invention areprobes and primers for the genes according to the invention, as well asnucleic acids which encode antigenic determinants of the gene productsaccording to the invention. Therefore, preferred embodiments includenucleic acids with preferably 10 to 50, or more preferably, 10 to 20consecutive nucleotides out of the disclosed sequences.

The term “nucleic acid” denotes a polynucleotide which can be, forexample, a DNA, RNA, or derivatized active DNA or RNA. DNA and mRNAmolecules are preferred, however.

The term “hybridize under stringent conditions” means that two nucleicacid fragments are capable of hybridization to one another understandard hybridization conditions described in Sambrook et al.,Molecular Cloning: A laboratory manual (1989) Cold Spring HarborLaboratory Press, New York, USA.

More specifically, “stringent conditions” as used herein refers tohybridization in 5.0×SSC, 5× Denhardt, 7% SDS, 0.5 M phosphate buffer pH7.0, 10% dextran sulfate and 100 μg/ml salmon sperm DNA at about 50°C.-68° C., followed by two washing steps with 1×SSC at 68° C. Inaddition, the temperature in the wash step can be increased from lowstringency conditions at room temperatures, about 22° C., to highstringency conditions at about 68° C.

The invention further comprises recombinant expression vectors which aresuitable for the expression of IIP-10, recombinant host cellstransfected with such expression vectors, as well as a process for therecombinant production of a protein which is encoded by the IIP-10 gene.

The invention further comprises synthetic and recombinant polypeptideswhich are encoded by the nucleic acids according to the invention, andpreferably encoded by the DNA sequence shown in SEQ ID NO:5 as well aspeptidomimetics based thereon. Such peptidomimetics have a high affinityfor cell membranes and are readily taken up by the cells.Peptidomimetics are preferably compounds derived from peptides andproteins, and are obtained by structural modification using unnaturalamino acids, conformational restraints, isosterical placement,cyclization, etc. They are based preferably on 24 or fewer, preferably20 or fewer, amino acids, a basis of approximately 12 amino acids beingparticularly preferred.

The polypeptides and peptidomimetics can be defined by theircorresponding DNA sequences and by the amino acid sequences derivedtherefrom. The isolated IIP polypeptide can occur in natural allelicvariations which differ from individual to individual. Such variationsof the amino acids are usually amino acid substitutions. However, theymay also be deletions, insertions or additions of amino acids to thetotal sequence leading to biologically active fragments. The IIP proteinaccording to the invention—depending, both in respect of the extent andtype, on the cell and cell type in which it is expressed—can be inglycosylated or non-glycosylated form. IIP-Polypeptides with tumoricidicand/or metastatic activity can be identified by a tumor progressionassay using mammalian cells expressing said polypeptides and measuringthe proliferation capacity and apoptosis in relation to mammalian cellsnot expressing said polypeptides. “Polypeptide with IIP-10 activity orIIP-10” therefore means proteins with minor amino acid variations butwith substantially the same activity as IIP-10. Substantially the samemeans that the activities are of the same biological properties and thepolypeptides show at least 75% homology (identity) in amino acidsequences with IIP-10. More preferably, the amino acid sequences are atleast 90% identical. Homology according to the invention can bedetermined with the aid of the computer programs Gap or BestFit(University of Wisconsin; Needleman and Wunsch, J. Biol. Chem. 48 (1970)443-453; Smith and Waterman, Adv. Appl. Math. 2 (1981) 482-489). OtherIIPs according to, and used by, the invention are in particular:

IIP-1

A cDNA encoding an IGF-1 receptor interacting protein which was namedIIP-1 (SEQ ID NO: 1) was isolated. The cDNA of IIP-1 codes for a newprotein of 333 aa with a calculated molecular weight of 35,727. IIP-1 isa glycine rich protein (13%). IIP-1 contains several N-myristoylationsites, PKC and Ck2 phosphorylation sites and two putative nuclearlocalization signals. A second isoform, IIP-I (p26), of 236 aa in lengthwith a calculated molecular weight of 26,071 was identified which wasgenerated most likely by alternative splicing (FIG. 3). Both isoformsbind to the IGF-1 receptor. cDNA sequences of IIP-I have been reportedpreviously (Database EMBL Nos. AF089818 and AF061263; DeVries, L., etal., Proc. Natl. Acad. Sci. USA 95 (1998) 12340-12345). Two overlappingcDNA clones (FIG. 4) were identified which show high homology to thehuman TIP-2 partial cDNA (GenBank accession number: AF028824) (Rousset,R., et al., Oncogene 16 (1998) 643-654) and were designated as IIP-1aand IIP-1b. The IIP-1a cDNA corresponds to nt 117 to 751 of TIP-2. TheIIP-1b cDNA shows besides TIP-2 sequences (nt 1 to 106) additional 5′sequences which are homologous to sequence Y2H35 of WO 97/27296 (nt 25to 158).

IIP-1a and IIP-1b both share the sequence coding for the PDZ domain ofTIP-2 (nt 156 to 410) which is a known protein-protein interactiondomain (Ponting, C. P., et al., BioEssays 19 (1997) 469-479). Bydeletion analysis the PDZ domain was determined as the essential andsufficient IGF-1 receptor binding domain of IIP-1 (FIG. 4). Furtheryeast two-hybrid analysis revealed that binding of the IIP-1 protein tothe IGF-1 receptor is specific for this receptor tyrosine kinase. Nointeraction was seen to the insulin receptor or Ros. Receptor tyrosinekinases of other families did not interact with IIP-1 (e.g. Met, Ret,Kit, Fms, Neu, EGF receptor). Thus, IIP-1 most likely is the firstinteraction protein shown to be specific for the IGF-1 receptor tyrosinekinase. IIP-1 also binds to the kinase inactive mutant of the IGF-1receptor.

IIP-2

IIP-2 was identified as a new binder of the cytoplasmic part of theIGF-1 receptor which corresponds to human APS (EMBL accession number:HSAB520). APS has been previously isolated in a yeast two-hybrid screenusing the oncogenic c-kit kinase domain as bait (Yokouchi, M., et al.,Oncogene 15 (1998) 7-15). IIP-2 interacts with the IGF-1 receptor in akinase dependent manner. Binding of IIP-2 was also observed to othermembers of the insulin receptor family (insulin receptor, Ros), but notto an unrelated receptor tyrosine kinase (Met). The region of IIP-2which was found to interact with the IGF-1 receptor corresponds to humanAPS (nt 1126 to 1674, EMBL Acc No. AB000520) contains the SH2 domain ofAPS (nt 1249 to 1545).

IIP-3

IIP-3 was isolated as a new IGF-1 receptor interacting protein and isidentical to PSM (GenBank accession number: AF020526). PSM is known as aPH and SH2 domain containing signal transduction protein which binds tothe activated insulin receptor (Riedel, H., et al., J. Biochem. 122(1997) 1105-1113). A variant of PSM has also been described (Riedel, H.,et al., J. Biochem. 122 (1997) 1105-1113). Binding of IIP-3 to the IGF-1receptor is dependent on tyrosyl phosphorylation of the receptor. A cDNAclone corresponding to nt 1862 to 2184 of the variant form of PSM wasidentified. The isolated cDNA clone turned out to code for the IGF-1receptor binding region. The SH2 domain of PSM (nt 1864 to 2148, EMBLAcc No. AF020526) is encoded by the sequence of the IIP-3 partial cDNAclone isolated.

IIP-4

IIP-4 was isolated as a new interacting protein of the cytoplasmicdomain of the IGF-1 receptor. IIP-4 corresponds to p59fyn, a src-liketyrosine kinase (EMBL accession number: MMU70324 and human fynEM_HUM1:HS66H14) (Cooke, M. P., and Perlmutter, R. M., New Biol. 1(1989) 66-74). IIP-4 binds in a kinase dependent manner to the IGF-1receptor and to several other receptor tyrosine kinases as to theinsulin receptor and Met. The region of IIP-4 interacting with the IGF-1receptor (nt 665 to 1044) contains the SH2 domain of p59fyn (EMBL AccNo. U70324).

IIP-5

IIP-5 was isolated as a new IGF-1 receptor interacting protein. IIP-5shows a high homology to the zinc finger protein Zfp38 (EMBL accessionnumber: MMZFPTA) and is at least 80% homologous to the correspondinghuman gene. Zfp-38 is known as a transcription factor (Chowdhury, K., etal., Mech. Dev. 39 (1992) 129-142). IIP-5 interacts exclusively with theactivated and phosphorylated IGF-1 receptor, but not with a kinaseinactive mutant. In addition to binding of IIP-5 to the IGF-1 receptorinteraction of IIP-5 with receptor tyrosine kinases of the insulinreceptor family (insulin receptor, Ros) was observed. IIP-5 does notbind to the more distantly related receptor tyrosine kinase Met.

One cDNA clone binding to the IGF-1 receptor which codes for nt 756 to1194 of Zfp38 (EMBL Acc No. MMZFPTA) and contains the first zinc finger(nt 1075 to 1158) was isolated. This domain is sufficient for binding tothe activated IGF-1 receptor.

IIP-6

IIP-6 was identified as a new IGF-1 receptor interacting protein. IIP-6shows weak similarity to the zinc finger domain of Zfp29 (EMBL accessionnumber: MMZFP29). Zfp29 consists of a N-terminal transcriptionalactivation domain and 14 C-terminal Cys²His² zinc fingers (Denny, P.,and Ashworth, A., Gene 106 (1991) 221-227). Binding of IIP-6 to theIGF-1 receptor depends on phosphorylation of the IGF-1 receptor kinase.IIP-6 also binds to the insulin receptor, but does not interact withMet. The region of IIP-6 found to interact with the IGF-1 receptor (SEQID NO:3, SEQ ID NO:4) contains two zinc finger domains of the Cys²His²type.

IIP-7

IIP-7 was isolated as a new IGF-1 receptor interacting protein whichcorresponds to Pax-3 (EMBL accession number: MMPAX3R and human Pax3EM-HUM2:S69369). Pax-3 is known as a DNA-binding protein being expressedduring early embryogenesis (Goulding, M. D., et al., EMBO J. 10 (1991)1135-1147). IIP-7 binds in a phosphorylation dependent manner to theIGF-1 receptor. IIP-7 also interacts with the insulin receptor and Met.A partial IIP-7 cDNA clone turned out to code for the IGF-1 receptorbinding domain of Pax3 (nt 815 to 1199, EMBL Acc No. MMPAX3R). Thisregion contains the Pax-3 paired damain octapeptide (nt 853 to 876) andthe paired-type homeodomain (nt 952 to 1134).

IIP-8

IIP-8 codes for the full-length cDNA of Grb7 (EMBL accession number:MMGRB7P, human Grb7 EM_HUM1:AB008789). Grb7, a PH domain and a SH3domain containg signal transduction protein, was first published as anEGF receptor-binding protein (Margolis, B. L., et al., Proc. Natl. Acad.Sci. USA 89 (1992) 8894-8898). IIP-8 does not interact with the kinaseinactive mutant of the IGF-1 receptor. Binding of IIP-8 to several otherreceptor tyrosine kinases (e.g. insulin receptor, Ros and Met) was alsoobserved.

IIP-9

IIP-9 was identified as a new IGF-1 receptor interaction protein. IIP-9is identical to nck-beta (EMBL Acc No. AF043260). Nck is a cytoplasmicsignal transduction protein consisting of SH2 and SH3 domains (Lehmann,J. M., et al., Nucleic Acids Res. 18 (1990) 1048). IIP-9 interacts withthe IGF-1 receptor in a phosphorylation dependent manner. nck binds tothe juxtamembrane region of the IGF-1 receptor. Apart from binding ofIIP-9 to the IGF-1 receptor, interaction with the insulin receptor butnot with Ros or Met was seen.

A preferred object of the invention are polypeptides that arehomologous, and more preferably, polypeptides that are substantiallyidentical to the polypeptides of SEQ ID NO:6 (IIP-10). Homology can beexamined by using the FastA algorithm described by Pearson, W. R.,Methods in Enzymology 183 (1990) 63-68, Academic Press, San Diego, US.By “substantially identical” is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for examplesubstitutions of one amino acid for another of the same class (e.g.valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative amino acid substitution, deletions or insertionslocated at positions of the amino acid sequence which do not destroy thebiological function of the polypeptide. This includes substitution ofalternative covalent peptide bonds in the polypeptide. By “polypeptide”is meant any chain of amino acids regardless of length orposttranslational modification (e.g., glycosylation or phosphorylation)and can be used interchangeably with the term “protein”.

According to the invention by “biologically active fragment” is meant afragment which can exert a physiological effect of the full-lengthnaturally-occurring protein (e.g., binding to its biological substrate,causing an antigenic response, etc.).

The invention also features fragments of the polypeptide according tothe invention which are antigenic. The term “antigenic” as used hereinrefers to fragments of the protein which can induce a specificimmunogenic response, e.g. an immunogenic response which yieldsantibodies which specifically bind to the protein according to theinvention. The fragments are preferably at least 8 amino acids, andpreferably up to 25 amino acids, in length. In one preferred embodiment,the fragments include the domain which is responsible for the binding ofthe IIPs to the IGF-1 receptor (i.e., the PDZ domain of IIP-1. By“domain” is meant the region of amino acids in a protein directlyinvolved in the interaction with its binding partner. PDZ domains areapproximately 90-residue repeats found in a number of proteinsimplicated in ion-, channel and receptor clustering and the linking ofreceptors to effector enzymes. Such PDZ are described in general byCabral, J. H., et al., Nature 382 (1996) 649-652.

The invention further comprises a method for producing a proteinaccording to the invention whose expression or activation is correlatedwith tumor proliferation, by expressing an exogenous DNA in prokaryoticor eukaryotic host cells and isolation of the desired protein orexpressing said exogeneous DNA in vivo for pharmaceutical means, whereinthe protein is coded preferably by a DNA sequence coding for IIP-10,preferably the DNA sequence shown in SEQ ID NO:5.

The polypeptides according to the invention can also be produced byrecombinant means, or synthetically. Non-glycosylated IIP-10 polypeptideis obtained when it is produced recombinantly in prokaryotes. With theaid of the nucleic acid sequences provided by the invention it ispossible to search for the IIP-10 gene or its variants in genomes of anydesired cells (e.g. apart from human cells, also in cells of othermammals), to identify these and to isolate the desired gene coding forthe IIP-10 protein. Such processes and suitable hybridization conditions(see also above, “stringent conditions”) are known to a person skilledin the art and are described, for example, by Sambrook et al., MolecularCloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press,New York, USA, and Hames, B. D., Higgins, S. G., Nucleic acidhybridisation—apractical approach (1985) IRL Press, Oxford, England. Inthis case the standard protocols described in these publications areusually used for the experiments.

The use of recombinant DNA technology enables the production of numerousactive IIP-10 derivatives. Such derivatives can, for example, bemodified in individual or several amino acids by substitution, deletionor addition. The derivatization can, for example, be carried out bymeans of site directed mutagenesis. Such variations can be easilycarried out by a person skilled in the art (J. Sambrook, B. D. Hames,loc. cit.). It merely has to be ensured by means of the below-mentionedtumor cell growth inhibition assay that the characteristic properties ofIIP-10 are preserved.

With the aid of such nucleic acids coding for an IIP-10 protein, theprotein according to the invention can be obtained in a reproduciblemanner and in large amounts. For expression in prokaryotic or eukaryoticorganisms, such as prokaryotic host cells or eukaryotic host cells, thenucleic acid is integrated into suitable expression vectors, accordingto methods familiar to a person skilled in the art. Such an expressionvector preferably contains a regulatable/inducible promoter. Theserecombinant vectors are then introduced for the expression into suitablehost cells such as, e.g., E. coli as a prokaryotic host cell orSaccharomyces cerevisiae, teratocarcinoma cell line PA-1 sc 9117(Büttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells,CHO or COS cells as eukaryotic host cells and the transformed ortransduced host cells are cultured under conditions which allowexpression of the heterologous gene. The isolation of the protein can becarried out according to known methods from the host cell or from theculture supernatant of the host cell. Such methods are described forexample by Ausubel I., Frederick M., Current Protocols in Mol. Biol.(1992), John Wiley and Sons, New York. Also in vitro reactivation of theprotein may be necessary if it is not found in soluble form in the cellculture.

The invention therefore in addition concerns a IIP polypeptide which isa product of prokaryotic or eukaryotic expression of an exogenous DNA.

The protein can be isolated from the cells or the culture supernatantand purified by chromatographic means, preferably by ion exchangechromatography, affinity chromatography and/or reverse phase HPLC.

IIP-10 can be purified after recombinant production by affinitychromatography using known protein purification techniques, includingimmunoprecipitation, gel filtration, ion exchange chromatography,chromatofocussing, isoelectric focussing, selective precipitation,electrophoresis, or the like.

Diagnostic Methods

The invention further comprises a method for detecting a nucleic acidmolecule encoding an IIP-gene, comprising incubating a sample (e.g.,body fluids such as blood, cell lysates) with a nucleic acid moleculeaccording to the invention and determining hybridization under stringentconditions of said nucleic acid molecule to a target nucleic acidmolecule for determination of presence of a nucleic acid molecule whichis said IIP gene and therefore a method for the identification of IGF-1Ractivation or inhibition in mammalian cells or body fluids.

Therefore the invention also includes a method for the detection of theproliferation potential of a tumor cell comprising

-   -   a) incubating a sample of body fluid of a patient suffering from        cancer, a sample of cancer cells, or a sample of a cell extract        or a cell culture supernatant of said cancer cells, whereby said        sample contains nucleic acids with a nucleic acid probe which is        selected from the group consisting of        -   (i) the nucleic acids shown in SEQ ID NOS:1, 3 or 5 or a            nucleic acid which is complementary thereto and        -   (ii) nucleic acids which hybridize under stringent            conditions with one of the nucleic acids from (i) and    -   b) detecting hybridization by means of a further binding partner        of the nucleic acid of the sample and/or the nucleic acid probe        or by X-ray radiography.

Hybridization between the probe and nucleic acids from the sampleindicates the presence of the RNA of such proteins. Such methods areknown to a person skilled in the art and are described, for example, inWO 89/06698, EP-A 0 200 362, U.S. Pat. No. 2,915,082, EP-A 0 063 879,EP-A 0 173 251, EP-A 0 128 018.

In a preferred embodiment of the invention the coding nucleic acid ofthe sample is amplified before the test, for example by means of theknown PCR technique. Usually a derivatized (labeled) nucleic acid probeis used within the framework of nucleic acid diagnostics. This probe iscontacted with a denatured DNA or RNA from the sample which is bound toa carrier and in this process the temperature, ionic strength, pH andother buffer conditions are selected—depending on the length andcomposition of the nucleic acid probe and the resulting meltingtemperature of the expected hybrid—such that the labeled DNA or RNA canbind to homologous DNA or RNA (hybridization see also Wahl, G. M., etal., Proc. Natl. Acad. Sci. USA 76 (1979) 3683-3687). Suitable carriersare membranes or carrier materials based on nitrocellulose (e.g.,Schleicher and Schüll, BA 85, Amersham Hybond, C.), strengthened orbound nitrocellulose in powder form or nylon membranes derivatized withvarious functional groups (e.g., nitro groups) (e.g., Schleicher andSchüll, Nytran; NEN, Gene Screen; Amersham Hybond M.; Pall Biodyne).

Hybridizing DNA or RNA is then detected by incubating the carrier withan antibody or antibody fragment after thorough washing and saturationto prevent unspecific binding. The antibody or the antibody fragment isdirected towards the substance incorporated during derivatization intothe nucleic acid probe. The antibody is in turn labeled. However, it isalso possible to use a directly labeled DNA. After incubation with theantibodies it is washed again in order to only detect specifically boundantibody conjugates. The determination is then carried out according toknown methods by means of the label on the antibody or the antibodyfragment.

The detection of the expression can be carried out for example as:

-   -   in situ hybridization with fixed whole cells, with fixed tissue        smears and isolated metaphase chromosomes,    -   colony hybridization (cells) and plaque hybridization (phages        and viruses),    -   Southern hybridization (DNA detection),    -   Northern hybridization (RNA detection),    -   serum analysis (e.g., cell type analysis of cells in the serum        by slot-blot analysis),    -   after amplification (e.g., PCR technique).

Preferably the nucleic acid probe is incubated with the nucleic acid ofthe sample and the hybridization is detected optionally by means of afurther binding partner for the nucleic acid of the sample and/or thenucleic acid probe.

The nucleic acids according to the invention are hence valuableprognostic markers in the diagnosis of the metastatic and progressionpotential of tumor cells of a patient.

Screening for Antagonists and Agonists of IIPs or Inhibitors

According to the invention antagonists of IIP-10 or inhibitors for theexpression of IIP (e.g., antisense nucleic acids) can be used to inhibittumor progression and cause massive apoptosis of tumor cells in vivo,preferably by somatic gene therapy. Therefore, the present inventionalso relates to methods of screening for potential therapeutics forcancer, diabetes, neurodegenerative disorders, bone diseases, to methodsof treatment for disease and to cell lines and animal models useful inscreening for and evaluating potentially useful therapies for suchdisease. Therefore another object of the invention are methods foridentifying compounds which have utility in the treatment of theafore-mentioned and related disorders. These methods include methods formodulating the expression of the polypeptides according to theinvention, methods for identifying compounds which can selectively bindto the proteins according to the invention and methods of identifyingcompounds which can modulate the activity of said polypeptides. Thesemethods may be conducted in vitro and in vivo and may employ thetransformed cell lines and transgenic animal models of the invention.

An antagonist of IIPs or an inhibitor of IIP is defined as a substanceor compound which inhibits the interaction between IGF-1R and IIP,preferably IIP-10. Therefore the biological activity of IGF-1R decreasesin the presence of such a compound. In general, screening procedures forIIP antagonists involve contacting candidate substances with IIP-bearinghost cells under conditions favorable for binding and measuring theextent of decreasing receptor mediated signaling (in the case of anantagonist). Such an antagonist is useful as a pharmaceutical agent foruse in tumor therapy. For the treatment of diabetes, neural diseases, orbone diseases, stimulation of the signaling pathway is required, i.e.,screening for agonists is useful.

IIP activation may be measured in several ways. Typically, theactivation is apparent by a change in cell physiology such as anincrease or decrease in growth rate or by a change in thedifferentiation state or by a change in cell metabolism which can bedetected in standard cell assays, for example MTT or XTT assays (RocheDiagnostics GmbH, DE).

The nucleic acids and proteins according to the invention couldtherefore also be used to identify and design drugs which interfere withthe interaction of IGF-1R and IIPs. For instance, a drug that interactswith one of the proteins could preferentially bind it instead ofallowing binding its natural counterpart. Any drug which could bind tothe IGF-1 receptor and, thereby, prevent binding of an IIP or, viceversa, bind to an IIP and, thereby, prevent binding of the IGF-1receptor. In both cases, signal transduction of the IGF-1 receptorsystem would be modulated (preferably inhibited). Screening drugs forthis facility occurs by establishing a competitive assay (assay standardin the art) between the test compound and interaction of IIP and theIGF-1 receptor and using purified protein or fragments with the sameproperties as the binding partners.

The protein according to the invention is suitable for use in an assayprocedure for the identification of compounds which modulate theactivity of the proteins according to the invention. Modulating theactivity as described herein includes the inhibition or activation ofthe protein and includes directly or indirectly affecting the normalregulation of said protein activity. Compounds which modulate theprotein activity include agonists, antagonists and compounds whichdirectly or indirectly affect the regulation of the activity of theprotein according to the invention. The protein according to theinvention may be obtained from both native and recombinant sources foruse in an assay procedure to identify modulators. In general, an assayprocedure to identify modulators will contain the IGF receptor, aprotein of the present invention, and a test compound or sample whichcontains a putative modulator of said protein activity. The testcompounds or samples may be tested directly on, for example, purifiedprotein of the invention, whether native or recombinant, subcellularfractions of cells producing said protein, whether native orrecombinant, and/or whole cells expressing said protein, whether nativeor recombinant. The test compound or sample may be added to the proteinaccording to the invention in the presence or absence of knownmodulators of said protein. The modulating activity of the test compoundor sample may be determined by, for example, analyzing the ability ofthe test compound or sample to bind to said protein, activate saidprotein, inhibit its activity, inhibit or enhance the binding of othercompounds to said protein, modifying receptor regulation or modifyingintracellular activity.

The identification of modulators of the protein activity are useful intreating disease states involving the protein activity. Other compoundsmay be useful for stimulating or inhibiting the activity of the proteinaccording to the invention. Such compounds could be of use in thetreatment of diseases in which activation or inactivation of the proteinaccording to the invention results in either cellular proliferation,cell death, non-proliferation, induction of cellular neoplastictransformations, or metastatic tumor growth and hence could be used inthe prevention and/or treatment of cancers such as, for example,prostate and breast cancer. The isolation and purification of a DNAmolecule encoding the protein according to the invention would be usefulfor establishing the tissue distribution of said protein as well asestablishing a process for identifying compounds which modulate theactivity of said protein and/or its expression.

Therefore a further embodiment of the invention is a method forscreening a compound that inhibits the interaction between IGF-1R andIIP-1, IIP-2, IIP3, IIP4, IIP5, IIP6, IIP7, IIP8, IIP9 or IIP-10,comprising

-   a) combining IGF-1R and the IIP polypeptide with a solution    containing a candidate compound such that the IGF-1R and the IIP    polypeptide are capable of forming a complex and-   b) determining the amount of complex relative to the predetermined    level of binding in the absence of said candidate compound and    therefrom evaluating the ability of said candidate compound to    inhibit binding of IGF-1R to the IIP polypeptide.

Such a screening assay is preferably performed as an ELISA assay wherebyIGF-1R or the IIP-polypeptide preferably IIP-1 or IIP-10, is bound on asolid phase. A further embodiment of the invention is a method for theproduction of a therapeutic agent for the treatment of carcinomas in apatient comprising combining a therapeutically effective amount of acompound which inhibits the interaction between IGF-1R and IIP inbiochemical and/or cellular assays to an extent of at least 50%.Biochemical assays are preferably ELISA-based assays or homogeneousassays. In the case of the ELISA system antibodies specific for the twobinding partners are used for detection of the complexes. In the case ofthe homogenous assay at least one binding partner is labeled withfluorophores which allows analysis of the complexes. Cellular assays arepreferably assays whereby tumor cells or cells transfected withexpression constructs of the IGF-1R and the respective binding proteinsare treated with or without drugs and complex formation between the twocomponents is then analyzed using standard cell assays.

A preferred embodiment of the invention is a method for the productionof a therapeutic agent for the treatment of carcinomas in a patientcomprising combining a pharmaceutically acceptable carrier with atherapeutically effective amount of a compound which inhibits theinteraction between IGF-1R and an IIP-polypeptide, preferably IIP-1 orIIP-10, in a cellular assay, whereby in said cellular assay tumor cellsor cells transfected with expression constructs of IGF-1R and of therespective IIP are treated with said compound, and complex formationbetween IGF-1R and said respective IIP is analyzed, and the extent ofsaid complex formation in the case of inhibition does not exceed 50%referred to 100% for complex formation without said compound in saidsame cellular assay.

A further embodiment of the invention is a method of treating a patientsuffering from a carcinoma with a therapeutically effective amount of acompound which inhibits the interaction between IGF-1R and theIIP-polypeptide, preferably IIP-1 or IIP-10, in a cellular assay,whereby in said cellular assay tumor cells or cells transfected withexpression constructs of IGF-1R and of the respective IIP are treatedwith said compound, and complex formation between IGF-1R and saidrespective IIP is analyzed, and the extent of said complex formation inthe case of inhibition does not exceed 50% referred to 100% for complexformation without said compound in said same cellular assay.

A further embodiment of the invention is an antibody against IIP-1 orIIP-10 according to the invention.

Antibodies were generated from the human, mouse, or rat polypeptides.Antibodies specifically recognizing IIP-1 or IIP-10 are encompassed bythe invention. Such antibodies are raised using standard immunologicaltechniques. Antibodies may be polyclonal or monoclonal or may beproduced recombinantly such as for a humanized antibody. An antibodyfragment which retains the ability to interact with IIP-1 or IIP-10 isalso provided. Such a fragment can be produced by proteolytic cleavageof a full-length antibody or produced by recombinant DNA procedures.Antibodies of the invention are useful in diagnostic and therapeuticapplications. They are used to detect and quantitate IIP-1 or IIP-10 inbiological samples, particularly tissue samples and body fluids. Theyare also used to modulate the activity of IIP-1 or IIP-10 by acting asan agonist or an antagonist.

The following examples, references, sequence listing and drawing areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

EXAMPLES Example 1

Isolation and Characterization of IGF-1R Binding Proteins

The yeast two-hybrid system (Fields, S., and Song, O., Nature 340 (1989)245-246) was used to isolate unknown cytosolic IGF-1 receptor bindingproteins. For screening a modified version of the yeast two-hybridsystem was used which allows interchain tyrosylphosphorylation of thereceptors in yeast. The yeast two-hybrid bait plasmid (BTM116-cpIGF-1receptor) was constructed by fusing the cytoplasmic domain of theβ-subunit of the IGF-1 receptor (nt 2923 to 4154) (Ullrich, A., et al.,EMBO J. 5 (1986) 2503-2512) to the LexA DNA-binding domain which formsdimers and mimics the situation of the activated wildtype receptor (cf.Weidner, M., et al., Nature 384 (1996) 173-176). By introducing aproline-glycine spacer between the LexA DNA-binding domain and thereceptor domain the ability of the bait to bind known substrates of theIGF-1 receptor was remarkably increased in comparision to other spaceramino acids (FIG. 1).

Alternatively a bait was constructed containing only the juxtamembraneor C-terminal region of the IGF-1 receptor (nt 2923 to 3051 or nt 3823to 4146) (Ullrich, A., et al., EMBO J. 5 (1986) 2503-2512) fused to thekinase domain of an unrelated, very potential receptor tyrosine kinase.Here the kinase domain of tpr met (nt 3456 to 4229) (GenBank accessionnumber: HSU19348) (FIG. 2) was used. In this way it is possible todelineate the region of the IGF-1 receptor which mediates binding todownstream effectors. The IGF-1 receptor bait plasmid was used to screenactivation domain cDNA libraries (e.g. VP16- or Gal4 based activationdomain) (cf. Weidner, M., et al., Nature 384 (1996) 173-176). The baitand prey plasmids were co-transfected into Saccharomyces cerevisiaestrain L40 containing a HIS3 and lacZ reporter gene. Library plasmidswere isolated from yeast colonies growing on histidine deficient medium,were sequenced and reintroduced into yeast strain L40. Byco-transfecting experiments with different test baits, i.e. BTM 116plasmids coding for a kinase inactive mutant of the IGF-1 receptor(L1033A) or the cytoplasmic domain of receptor tyrosine kinases of theinsulin receptor family (insulin receptor, Ros) and of unrelatedreceptor tyrosine kinase families (Met, EGF receptor, Kit, Fms, Neu) thespecificity of the putative bait-prey interactions was evaluated.Several cDNAs were identified which code for previously unknown IGF-1receptor interacting proteins (IIPs). In addition binding domains ofknown substrates of the IGF-1 receptor such as the C-terminal SH2 domainof p85PI3K and the SH2 domain of Grb10 were found. The results are shownin Table 1.

TABLE 1 IIP wt IGF-1R mu IGF-1R IR Ros Met IIP-1 + + − − − IIP-2 + − + +− IIP-3 + − + + + IIP-4 + − + nd + IIP-5 + − + + − IIP-6 + − + nd −IIP-7 + − + nd + IIP-8 + − + + + IIP-9 + − + − − IIP-10 + − − nd nd

Delineation of the binding specificity of the IIPs with respect todifferent receptor tyrosine kinases tested in the yeast two-hybridsystem. Yeast cells were cotransfected with a LexA fusion constructcoding for the different receptor tyrosine kinases and an activationplasmid coding for the different IIPs fused to the VP16 activationdomain. Interaction between the IIPs and the different receptor tyrosinekinases was analyzed by monitoring growth of yeast transfectants platedout on histidine deficient medium and incubated for 3d at 30° C. (wtIGF-1R, kinase active IGF-1 receptor; mu IGF-1R, kinase inactive mutantIGF-1 receptor; IR, insulin receptor; Ros, Ros receptor tyrosine kinase;Met, Met receptor tyrosine kinase; +, growth of yeast transfectantswithin 3 days larger than 1 mm in diameter; −, no detected growth; nd,not determined).

Example 2

Assay Systems

A) In-vitro/biochemical Assays:

ELISA-based assay/homogenous assay

IGF-1R and the binding proteins (IIPs) are expressed with or withoutTag-epitopes in E.coli or eucaryotic cells and purified to homogeneity.Interaction of IGF-1R and the respective binding proteins are analyzedin the presence or absence of drugs. Compounds which either inhibit orpromote binding of IGF-1R and the respective binding proteins areselected. In the case of the ELISA system antibodies specific for thetwo binding partners are used for detection of the complexes. In thecase of the homogenous assay at least one binding partner is labeledwith fluorophores which allows analysis of the complexes. Alternatively,anti-Tag-antibodies are used to monitor interaction.

B) Cellular Assays

Tumor cells or cells transfected with expression constructs of theIGF-1R and the respective binding proteins are treated with or withoutdrugs and complex formation between the two components is then analyzedusing standard assays.

Example 3

cDNA Cloning of IIP-1 and IIP-10 (and RT-PCR-assay)

The nucleotide sequence of full length IIP-1 was determined bysequencing of the partial cDNA clones of IIP-1 and (IIP-1a, IIP-1b) andby using database information (ESTs). cDNA cloning of full length IIP-1was performed by RT PCR on total RNA isolated from a MCF7_(ADR)breastcell line. PT PCR with two oligonucleotide primers: TIP2c-s (SEQ IDNO:7) and TIP2b-r (SEQ ID NO:8) resulted in amplification of two DNAfragments of 1.0 kb (IIP-1) and 0.7 kb (IIP-1(p26)).

The nucleotide sequence of full length IIP-10 was determined bysequencing of the partial cDNA clone of IIP-10 and by using databaseinformation (ESTs). cDNA cloning of IIP-10 was performed on total RNAisolated from the colon cancer cell line SW480. RT PCR with twooligonucleotide primers: Hethy-s (SEQ ID NO:9) and Hcthy-r (SEQ ID NO:10) resulted in amplification of a cDNA fragment of 676bp (IIP-10). DNAsequencing was performed using the dideoxynucleotide chain terminationmethod on an ABI 373A sequencer using the Ampli Taq® FSDideoxyterminator kit (Perkin Elmer, Foster City, Calif.). Comparison ofthe cDNA and deduced protein sequences was performed using AdvancedBlast Search (Altschul, S. F., et al., J. Mol. Biol. 215 (1990) 403-410;Altschul, S. F., et al., Nucleic Acids Res. 25 (1997) 3389-3402).

Example 4

Western Blot Analysis of IIP-1 and IIP-10

Total cell lysates were prepared in a buffer containing 50 mM Tris pH8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, and 1 mMEDTA and cleared by centrifugation for 15 min at 4° C. The proteinconcentration of the supernatants was measured using the Micro BCAProtein Assay kit (Pierce Chemical Co., Rockford, Ill.) according to themanufacturer's manual. IGF-1 receptors were immunoprecipitated usinganti-IGF-1 receptor antibodies (Santa Cruz). Proteins were fractionatedby SDS-PAGE and electrophoretically transferred to nitrocellulosefilters. Nitrocellulose filters were preincubated with 10% (w/v)fat-free milk powder in 20 mM Tris pH 7.5, 150 mM NaCl, 0.2% Tween-20.Binding of a mouse monoclonal antibody directed against the flag epitopewas detected by horseradish peroxidase-labeled goat-anti-mouse IgGantiserum (Biorad, Munich, DE) and visualized using an enhancedchemoluminescence detection system, ECL™ (Amersham, Braunschweig, DE).

Example 5

Overexpression of IIP-1 to IIP-10 in Mammalian Cells byLiposome-mediated Transfection

The cDNAs for IIP-1 to -10 were cloned into the NotI site of pBATflag orpcDNA3flag (Weidner, K. M., et al., Nature 384 (1996) 173-176); Behrens,J., et al., Nature 382 (1996) 638-642; Behrens, J., et al., Science 280(1998) 596-599). NIH3T3 cells or other recipient cells were transfectedwith pcDNAflagIIP-1 to -10 or alternatively with pBATflag IIP-1 to -10using FuGENE6 (Roche Biochemicals) as transfection agent. Cells wereselected in 0.4 mg/ml G418. Single clones were picked and analzyed forexpression of IIP-1 to -10 and functionally characterized with respectto proliferation. Northern blot analysis

Human and murine mRNA multiple tissue Northern blots were purchased fromClontech (Palo Alto, Calif., US). A cDNA probe spanning IIP-10nt343-nt676 of the coding region was labeled with DIG-dUTP using the PCRDIG Labeling Mix (Roche Diagnostics GmbH, DE). A digoxygenin labeledactin RNA probe was purchased from Roche Diagnostics GmbH, DE.Hybridization was performed using the DIG EasyHyb hybridization solution(Roche Diagnostics GmbH, DE). IIP-10 mRNA was detected with DIG-specificantibodies conjugated to alkaline phosphatase and the CSPD substrate(Roche Diagnostics GmbH, DE).

Example 6

Detection of mRNA in Cancer Cells

In order to detect whether proteins are expressed in cancer cells whichare coded by nucleic acids which hybridize with SEQ ID NO:1 or SEQ IDNO:5 or the complementary sequence and consequently whether MRNA ispresent, it is possible on the one hand to carry out the establishedmethods of nucleic acid hybridization such as Northern hybridization,in-situ hybridization, dot or slot hybridization and diagnostictechniques derived therefrom (Sambrook et al., Molecular Cloning: Alaboratory manual (1989) Cold Spring Harbor Laboratory Press, New York,USA; Hames, B. D., Higgins, S. G., Nucleic acid hybridisation—apractical approach (1985) IRL Press, Oxford, England; WO 89/06698; EP-A0 200 362; EP-A 0 063 879; EP-A 0 173 251; EP-A 0 128 018). On the otherhand it is possible to use methods from the diverse repertoire ofamplification techniques using specific primers (PCR Protocols—A Guideto Methods and Applications (1990), publ. M. A. Innis, D. H. Gelfand, J.J. Sninsky, T. J. White, Academic Press Inc.; PCR—A Practical Approach(1991), publ. M. J. McPherson, P. Quirke, G. R. Taylor, IRL Press).

The RNA for this is isolated from the cancer tissue by the method ofChomcszynski and Sacchi, Anal. Biochem. 162 (1987) 156-159.20 μg totalRNA was separated on a 1.2% agarose formaldehyde gel and transferredonto nylon membranes (Amersham, Braunschweig, DE) by standard methods(Sambrook et al., Molecular Cloning: A laboratory manual (1989) ColdSpring Harbor Laboratory Press, New York, USA. The DNA sequence SEQ IDNO:1 or SEQ ID NO:5 was radioactively labeled as probes (Feinberg, A.P., and Vogelstein, B., Anal. Biochem. 137 (1984) 266-267). Thehybridization was carried out at 68° C. in 5× SSC, 5× Denhardt, 7%SDS/0.5 M phosphate buffer pH 7.0, 10% dextran sulfate and 100 μg/mlsalmon sperm DNA. Subsequently the membranes were washed twice for onehour each time in 1×SSC at 68° C. and then exposed to X-ray film.

Example 7

Procedure for Identification of Modulators of the Activity of theProtein According to the Invention

The expression vector of Example 5 (either for IIP-1 or IIP-10 10 μg/10⁶cells) is transferred into NIH 3T3 cells by standard methods known inthe art (Sambrook et al.). Cells which have taken up the vector areidentified by their ability to grow in the presence of the selection orunder selective conditions (0.4 mg/ml G418). Cells which express DNAencoding IIP produce RNA which is detected by Northern blot analysis asdescribed in Example 5. Alternatively, cells expressing the protein areidentified by identification of the protein by Western blot analysisusing the antibodies described in Example 4. Cells which express theprotein from the expression vector will display an altered morphologyand/or enhanced growth properties.

Cells which express the protein and display one or more of the alteredproperties described above are cultured with and without a putativemodulator compound. By screening of chemical and natural libraries, suchcompounds can be identified using high throughput cellular assaysmonitoring cell growth (cell proliferation assays using as chromogenicsubstrates the tetrazolium salts WST-1, MTT, or XTT, or a cell deathdetection ELISA using bromodesoxyuridine (BrdU); cf. Boehringer MannheimGmbH, Apoptosis and Cell Proliferation, 2^(nd) edition, 1998, pp.70-84).

The modulator compound will cause an increase or a decrease in thecellular response to the IIP protein activity and will be either anactivator or an inhibitor of IGF-receptor function, respectively.

Alternatively, putative modulators are added to cultures of tumor cells,and the cells display an altered morphology and/or display reduced orenhanced growth properties. A putative modulator compound is added tothe cells with and without IIP protein and a cellular response ismeasured by direct observation of morphological characteristics of thecells and/or the cells are monitored for their growth properties. Themodulator compound will cause an increase or a decrease in the cellularresponse to IIP protein and will be either an activator or an inhibitorof IGF-1 receptor activity, respectively.

List of References

-   Altschul, S. F., et al., J. Mol. Biol. 215 (1990) 403-410-   Altschul, S. F., et al., Nucleic Acids Res. 25 (1997) 3389-3402-   Ausubel I., Frederick M., Current Protocols in Mol Biol. (1992),    John Wiley and Sons, New York-   Weidner, K. M., et al., Nature 384 (1996) 173-176); Behrens, J., et    al., Nature 382 (1996) 638-642-   Behrens, J., et al., Science 280 (1998) 596-599-   Boehringer Mannheim GmbH, Apoptosis and Cell Proliferation, 2nd    edition, 1998, pp. 70-84-   Büttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583-   Cabral, J. H., et al., Nature 382 (1996) 649-652-   Chomcszynski and Sacchi, Anal. Biochem. 162 (1987) 156-159-   Chowdhury, K., et al., Mech. Dev. 39 (1992) 129-142-   Cooke, M. P., and Perlmutter, R. M., New Biol. 1 (1989) 66-74-   Database EMBL Nos. AF089818 and AF061263-   Denny, P., and Ashworth, A., Gene 106 (1991) 221-227-   DeVries, L., et al., Proc. Natl. Acad. Sci. USA 95 (1998)    12340-12345-   Dey, R. B., et al., Mol. Endocrinol. 10 (1996) 631-641-   EP-A 0 063 879-   EP-A 0 128 018-   EP-A 0 173 251-   EP-A 0 200 362-   Feinberg, A. P., and Vogelstein, B., Anal. Biochem. 137 (1984)    266-267-   Fields, S., and Song, O., Nature 340 (1989) 245-246-   Goulding, M. D., et al., EMBO J. 10 (1991) 1135-1147-   Hames, B. D., Higgins, S. G., Nucleic acid hybridisation—a practical    approach (1985) IRL Press, Oxford, England-   Lehmann, J. M., et al., Nucleic Acids Res. 18 (1990) 1048)-   Margolis, B. L., et al., Proc. Natl. Acad. Sci. USA 89 (1992)    8894-8898-   Needleman and Wunsch, J. Biol. Chem. 48 (1970) 443-453-   PCR—A Practical Approach (1991), publ. M. J. McPherson, P.    Quirke, G. R. Taylor, IRL Press-   Pearson, W. R., Methods in Enzymology 183 (1990) 63-68, Academic    Press, San Diego, US-   Ponting, C. P., et al., BioEssays 19 (1997) 469-479-   PCR Protocols—A Guide to Methods and Applications (1990),    publ. M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White,    Academic Press Inc.-   Riedel, H., et al., J. Biochem. 122 (1997) 1105-1113-   Rousset, R., et al., Oncogene 16 (1998) 643-654-   Sambrook et al., Molecular Cloning: A laboratory manual (1989) Cold    Spring Harbor Laboratory Press, New York, USA-   Smith and Waterman, Adv. Appl. Math. 2 (1981) 482-489-   Ullrich, A., et al., EMBO J. 5 (1986) 2503-2512-   U.S. Pat. No. 2,915,082-   Wahl, G. M., et al., Proc. Natl. Acad. Sci. USA 76 (1979) 3683-3687-   Weidner, K. M., et al., Nature 384 (1'996) 173-176-   WO 97/27296-   WO 89/06698-   WO 95/14772-   Yokouchi, M., et al., Oncogene 15 (1998) 7-15

1. A method for the detection of a nucleic acid molecule encoding anIGF-1 receptor interacting protein comprising; a) incubating a samplewith a nucleic acid probe that is selected from the group consisting of:(i) a nucleic acid probe having the sequence SEQ ID No: 5 or a nucleicacid which is at least 90% identical thereto; and (ii) a nucleic acidprobe that though not identical to the nucleic acid probes from (i),but, due to the degeneracy of the genetic code encode a polypeptidehaving the amino acid sequence of the polypeptides encoded by nucleicacid sequences of (i); and b) detecting whether hybridization hasoccurred.
 2. The method of claim 1 wherein said sample is selected fromthe group consisting of body fluid of a patient suffering from cancer;tumor cells; a tumor cell extract; and a cell culture supematant of saidtumor cells.
 3. The method of claim 1 wherein the nucleic acid to bedetected is amplified before the detection.
 4. A method for thedetection of a nucleic acid molecule encoding an IGF-1 receptorinteracting protein comprising: a) incubating a sample with a nucleicacid probe that is selected from the group consisting of: (i) a nucleicacid probe having the sequence SEQ ID No:5; and (ii) a nucleic acidprobe that though not identical to the nucleic acid probe of (i), but,due to the degeneracy of the genetic code encodes a polypeptide havingthe amino acid sequence of the polypeptide encoded by the nucleic acidsequence of (i); and b) detecting whether hybridization has occurred.